The invention refers to a modified agrin-fragment for use as a medicament. Additionally the invention refers to the use of the modified agrin-fragment for the manufacturing of a medicament for the treatment of different especially muscular or motor neuron diseases but also for the treatment of further neurotrypsin related diseases. Furthermore the invention refers to the use of the modified agrin-fragment in the treatment of different diseases. Finally the invention relates to a pharmaceutical composition comprising the modified agrin-fragment and to a special modified agrin fragment.
Aging people of 60 years or more face an inexorable decline of lean body mass. Because this is commonplace it was not held worth while to investigate the underlying mechanism for a long time. Currently the age-related decline in lean body mass are referred to as sarcopenia or frailty.
Sarcopenia and frailty are both highly relevant entities with regard to functionality and independence in the elderly. Sarcopenia is regarded as the degenerative age-related decline in muscle mass and strength leading at its late stage to frailty and disability. The muscle mass declining rate of sarcopenia patients is significantly faster than of people not affected with the disease. Sarcopenia is not a static condition but aggravating with aging. Because of its slow development, people usually are unaware of sarcopenia until having reached already an advanced stage.
Sarcopenic muscle mass is characterized by a continuously shrinking number of muscle fibers. The fiber size is more heterogeneous, and fiber type grouping is observed. Fiber type grouping is thought to be caused by repeated denervation/reinnervation. In addition, aged muscle is characterized by infiltration of fat and connective tissue. All this causes a decline in muscle strength and speed of movement. The age-related changes observed in many muscles are thought to be caused to some extent by age-related changes in the innervation of their muscle fibers. During aging a significant and progressive reduction in the estimated number of functioning motor units (MU) is observed. Similarly, an apparent muscle strength decline associated with a substantial loss of MUs is observed in individuals afflicted with motoneuron diseases such as amyotrophic lateral sclerosis (ALS) or less severe forms of spinal muscle atrophy (SMA).
Cycles of denervation/reinnervation of muscle fibers requires synaptic plasticity. The process of synaptic plasticity is not yet understood in detail. However, in the past years some key players in the formation and maintenance of neuromuscular junctions (NMJ's) were identified (Song and Balice-Gordon, 2008). The well characterized protein Agrin (Bezakova and Ruegg, 2003) plays a pivotal role in the synapse-formation process by assisting formation and maintenance of the postsynaptic apparatus of developing NMJs.
Agrin is a large heparan proteoglycan with a molecular weight of 400-600 kDa. (Database accession number NP—940978). The protein core consists of about 2000 amino acids and its mass is about 225 kDa. It is a multidomain protein composed of 9 K (kunitz-type) domains, 2 LE (laminin-EGF-like) domains, one SEA (sperm protein, enterokinase and agrin) domain, 4 EG (epidermal growth factor-like) domains and 3 LG (laminin globular) domains (FIG. 1). Agrin is a very important protein and agrin deficient mice die at birth due to respiratory failure. This is caused by the fact that agrin is strictly required for the proper innervation of muscle fibers and that these mice are not able to build proper NMJ's.
Agrin exists in several splice variants and can be expressed as a secreted protein, containing the N-terminal NtA (N-terminal agrin) domain, which is the most abundant form of agrin and the predominant form expressed in motor neurons. It is produced in the soma of the neurons, transported down the axon and released from the axon ending of the motor nerve into the synaptic cleft of the NMJ. Here it acts as an agonist of LRP4 and may also become a component of the basal lamina. In the CNS, most agrin is expressed as a type-II transmembrane protein by alternative splicing at the N-terminus lacking the N-terminal NtA domain (Bezakova and Ruegg, 2003).
The serine/threonine (S/T) rich segments in agrin are responsible for a high degree of glycosylation, containing several glycosylation and glucosaminoglycan attachment sites giving rise to the big mass of the proteoglucan. The C-terminal, 75 kDa moiety of agrin starting with the first EG domain is required for full activity in acetylcholine receptor (AChR) clustering activity on muscle cells, although the most C-terminal 20 kDa fragment is sufficient to induce AChR aggregation (Bezakova and Ruegg, 2003). Several binding sites for interaction partners of agrin, including α-dystroglycan, heparin, some integrins and LRP4 are mapped to the C-terminal region. The large heparansulfate side chains are binding sites for heparin binding proteins, e.g some growth factors.
In the C-terminal part of human agrin, there are 2 alternative splice sites y and z At the y-site, there may be inserts of 0, 4, 17 or 21 (4+17) amino acids and at the z site there may be inserts of 0, 8, 11 or 19 (8+11) amino acids. The function of the four inserted amino acids in the y-site is to create a heparin binding site. Motor neurons express predominantly y4 agrin. The most important splice site of agrin in respect of NMJ maturation is the z-site, giving agrin the ability to be active as an acetylcholine-receptor clustering agent. It is well known that full length agrin containing the insertion of 8 amino acids at the z-site in presence of the 4 amino acid insert in splice site y (y4z8) generates an agrin variant with a half maximal AChR clustering activity of 35 pM in cultured myotube clustering assays. The insertion of 11 amino acids give rise to a half maximal AChR clustering activity of 5 nM while the 19 amino acid insertion results in a half maximal AChR clustering activity of 110 pM. Agrin without an insertion at this site is not active in clustering acetylcholine-receptors on the in-vitro cultured myotubes (Bezakova and Ruegg, 2003). Thus, the most active form of agrin in the clustering assay is the y4z8 variant, which is expressed by motor neurons.
A ˜40 kDa C-terminal fragment of agrin (y4z8) containing the LG2, EG4 and the LG3 domains was found to be active in AChR clustering with an EC50 of 130 pM in the AchR clustering activity while shorter fragments have only lower activities. The C-terminal LG3 domain with the z8 insertion exhibits a half maximal AChR clustering activity of only 13 nM, which is a factor 100 fold lower than the 40 kDa fragment (Bezakova and Ruegg, 2003).
During the development and maturation of the NMJ, agrin is a key player of molecules involved in the clustering of acetycholine-receptors. While NMJ's are destabilized by the neurotransmitter acetyl choline, agrin, which is secreted by the motor neuron, stabilizes and increases the clusters of the AChR's via phosphorylation of MuSK, a membrane bound receptor tyrosine kinase. The interaction of Agrin with MuSK is postulated to be mediated via LRP4, a low-density lipoprotein receptor (LDLR)-related protein. It was found that agrin (y4z8) has a ˜10 fold higher affinity to LRP4 than agrin (y4z0) giving rise to the differential AChR clustering activity of the different agrin splice variants observed in the in-vitro cultured myotube assays. Upon agrin-binding, LRP4 causes self-phosphorylation of MuSK, which then activates the signal cascade for the expression and clustering of acetylcholine receptors.
Neurotrypsin is found in spinal cord extracts and is produced by motor neurons. Neurotrypsin is a secreted, trypsin like serine protease which is produced by CNS neurons, as well as by motor neurons (Stephan et al., 2008). Neurotrypsin consists of an N-terminal non catalytic part, containing a proline rich basic (PB) segment, a kringle (KR) domain, 4 scavenger receptor cysteine-rich (SRCR) domains, and a C-terminal protease domain (Gschwend et al., 1997). In human, a 4 bp deletion in exon 7 leads to a severe nonsyndromic mental retardation. The mutation leads to a truncated form of neurotrypsin lacking the protease domain, thus disabling the proteolytic function of the enzyme.
At present, agrin is the only known target of neurotrypsin. Neurotrypsin cleaves agrin at 2 distinct sites called α- and β-site. (FIG. 1). The α-site is located N-terminal from the SEA domain and the β-site is placed in front of the LG3 domain of agrin. Cleavage at the α-site generates a ˜100 kDa C-terminal agrin fragment running at ˜130 kDa in a 4-12% bis-tris SDS gel. Cleavage at the β-site liberates the C-terminal LG3 domain running at ˜22 kDa in the gel (Molinari et al., 2002; Reif et al., 2007). All C-terminal fragments could be detected in brain extracts and spinal cord extracts of mice (Stephan et al., 2008). In neurotrypsin knock out mice, none of the fragments could be detected. As a consequence, neurotrypsin seems to be the only protease which cleaves agrin in significant amounts at the two cleavage sites. Cleavage of agrin by neurotrypsin contributes to neuromuscular plasticity and remodelling
It was found that neurotrypsin (NT) overexpressing mice, so-called sarcopenia mice (muslik, M491S) (Stephan et al., 2008), show an early onset of sarcopenia. Detailed findings from systematic analysis of the sarcopenia mice demonstrate that a good correlation exists to human data. U.S. Ser. No. 12/007,928 gives a description of the animal model. Briefly the sarcopenia mice (muslik M491S) show:                (i) that nerve fibres of mouse diaphragms grow towards the individual endplates,        (ii) fragmented endplates on the diaphragm muscles, eventually leading to the disappearance of individual endplates, i.e. a loss of pre- and postsynaptic sites,        (iii) reduced fibre number and decreased homogeneity of fibre size in mouse soleus muscles in neurotrypsin overexpressing animals. The sarcopenia mice has a reduced fibre number (about 30%) and an increased inhomogeneity of fibre sizes.        
In WO 97/21811 it is proposed to use agrin or agrin fragments in methods of treatment of a disease that affects muscle. However, such attempts have up to date not shown to be successful
The object of the present invention is to provide a therapy for different muscular disorders involving a reduction of functional NMJs and/or motor-units (MU).